Antigen Unmasking Is Required to Clinically Assess Levels and Localisation Patterns of Phospholipase C Zeta in Human Sperm

Mammalian oocyte activation is initiated by intracellular calcium (Ca2+) oscillations, driven by the testis-specific phospholipase C zeta (PLC zeta). Sperm PLC zeta analysis represents a diagnostic measure of sperm fertilisation capacity. The application of antigen unmasking/retrieval (AUM) generally enhanced the visualisation efficacy of PLC zeta in mammalian sperm, but differentially affected the PLC zeta profiles in sperm from different human males. It is unclear whether AUM affects the diagnosis of PLC zeta in human sperm. Herein, we examined whether the application of AUM affected the correlation of PLC zeta profiles with sperm parameters and fertilisation capacity. PLC zeta fluorescence levels and localisation patterns were examined within the sperm of males undergoing fertility treatment (55 patients aged 29-53) using immunofluorescence in the absence/presence of AUM. The changes in PLC zeta profiles following AUM were examined in relation to sperm health and fertilisation outcome. AUM enhanced the observable levels and specific localisation patterns of PLC zeta in relation to both optimal sperm parameters and fertilisation outcome, without which significant differences were not observed. The extent of the change in levels and localisation ratios of PLC zeta was also affected to a larger degree in terms of the optimal parameters of sperm fertility and fertilisation capacity by AUM. Collectively, AUM was essential to accurately assesses PLC zeta in human sperm in both scientific and clinical contexts.